Project Summary In eukaryotic cells, nucleosomes prevent efficient binding of transcription factors and transcription machinery to their genomic targets. A special group of transcription factors called ?pioneer factors? can invade compact chromosome and remodel nucleosomes near their binding sites, allowing the access of transcription factors and triggering their transcriptional competency. Pioneer factors play critical roles in gene regulation and cell differentiation. Mutation or mis-regulation of pioneer factors are associated with developmental diseases and many types of human cancer. Despite their essential functions, the mechanism underlying the pioneer activity is unclear. Our long-term goal is to develop a detailed mechanistic understanding of how pioneer factors access their nucleosomal binding sites, disrupt histone-DNA contacts, and activate gene expression. To achieve this goal, our overall objective in this proposal is to quantitatively measure the binding, nucleosome-depleting, and gene regulatory activities of pioneer factors under a variety of genetic and cellular conditions. We will perform these measurements into three aims. At the population level, we will determine the steady-state nucleosome-depleting activities among genome-wide TFs to understand what kind of TFs can serve as pioneers and how their nucleosome-depleting activities are regulated in vivo (aim 1). At the single cell level, we will evaluate the gene regulatory activities of pioneer factors and their cell-to-cell variability (aim 2). At the single molecule level, we will directly measure the kinetics of pioneer factor binding and invasion of the nucleosome substrates in vitro (aim 3). Completion of these aims is expected to advance the fundamental knowledge of pioneer factors and their role in chromosome opening and gene regulation. It will also contribute to future dissection of the functional roles of pioneer factors in cancer cells and the development of novel therapies to combat their dysfunction.